PROJECT SUMMARY The enterovirus genus includes many medically and socioeconomically important human pathogens such as poliovirus, coxsackievirus, EV-A71, EV-D68, and rhinoviruses. Non-polio enteroviruses (NPEV) such as EV-A71 and EV-D68 are of particular concern as they are causative pathogens for hand, foot, and mouth disease (HFMD), moderate to severe respiratory illness and, in rare cases, central nervous system (CNS) infections in humans. Both EV-A71 and EV-D68 are listed among the NIH NIAID priority pathogens. Despite a significant disease burden, no approved antiviral drugs are currently available for the prevention and treatment of EV-A71 and EV-D68 infection. Accordingly, the goal of the proposed research is to identify viral 2A protease inhibitors as potent EV-A71 and EV-D68 antivirals through high-throughput screening (HTS). The EV-A71 and EV-D68 2A proteases play an essential role in viral replication by cleaving the viral polyprotein after viral transcription, initiating viral replication. The EV-A71 and EV-D68 2A proteases represent unexplored novel antiviral drug targets?only three weak or promiscuous EV-A71 2Apro inhibitors have been reported and there is no EV-D68 2Apro inhibitor in the literature. To identify EV-A71 and EV-D68 2Apro inhibitors through HTS, we developed fluorescence resonance energy transfer (FRET)-based enzymatic assays for both the EV-A71 and EV?D68 2Apro proteins. The EV-D68 2Apro FRET assay was subsequently used to conduct a pilot screening against the Selleckchem Bioactive compound library (1902 compounds), leading to the identification of telaprevir (VX-950) as the first EV-D68 2Apro inhibitor with an IC50 of 0.2 M. In secondary cell culture assays, telaprevir showed sub- to low micromolar potency against several contemporary human EV-D68 strains in different human cell lines. The success of the pilot screening suggests that it is feasible to identify 2Apro inhibitors as EV antivirals through FRET-based HTS. As EV-A71 and EV-D68 cause both respiratory infection and, in rare cases, neurological infection, there is a need for both blood-brain barrier (BBB)-impermeable and BBB-permeable 2Apro inhibitors. BBB-impermeable 2Apro inhibitors are the most needed for the treatment of respiratory infection as well as the prevention of viral spread to the CNS. In contrast, BBB-permeable 2Apro inhibitors are needed for the treatment of neurological infection when virus has spread to the spinal cord through viremia. In addition, as the EV-A71 and EV-D68 2A proteases share ~60% sequence similarity, we expect to identify inhibitors that target either EV-A71 2Apro or EV- D68 2Apro or both by conducting two FRET-based enzymatic HTS assays?one for EV-A71 2Apro and one for EV- D68 2Apro. The expected outcome of this project is the identification of first-in-class EV-A71 and EV-D68 2Apro inhibitors with validated mechanisms of action and drug-like properties. The proposed study is significant because it represents a step forward towards addressing the unmet medical need for EV-A71 and EV-D68 antivirals.